Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Resolution of optical isomers or purification of organic...
Reexamination Certificate
2000-10-16
2003-04-15
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Resolution of optical isomers or purification of organic...
Reexamination Certificate
active
06548293
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to enzymatic processes for the resolution of enantiomeric mixtures of &bgr;-lactams useful in the preparation of taxanes.
The taxane family of terpenes, of which taxol and docetaxel are members, has attracted considerable interest in both the biological and chemical arts. Such taxanes may be prepared through a variety of semi-synthetic routes. In one, a &bgr;-lactam is coupled to a derivative of 10-deacetylbaccatin III to form a sidechain at the C-13 position of the derivative. As the stereochemistry of these taxanes may affect their pharmaceutical activity, methods allowing efficient stereospecific preparation of the intermediate &bgr;-lactam, as well as the final taxane products, have been the subject of investigation.
Brieva et al. (Brieva, R.; Crich, J. Z. and Sih, C. J.,
J. Org. Chem
. 1993,58, 1068) reported that racemic &bgr;-lactam underwent selective kinetic hydrolysis with several Pseudomonas lipases and two penicillinases. Pseudomonas lipases used by Brieva et al. include P-30, AK and K-10.
Similarly, Patel reported in U.S. Pat. No. 5,879,929, that enantiomeric mixtures of certain &bgr;-lactams and, in particular, racemic mixtures of certain &bgr;-lactams, can be resolved by a stereoselective hydrolysis using a variety of lipases and enzymes. Lipases identified by Patel include Amano PS-30 (
Pseudomonas cepacia
), Amano GC-20 (
Geotrichum candidum
), Amano APF (
Aspergillus niger
), Amano AK (Pseudomonas sp.),
Pseudomonas fluorescens
lipase (Biocatalyst Ltd.), Amano Lipase P-30 (Pseudomonas sp.), Amano P (
Pseudomonas fluorescens
), Amano AY-30 (
Candida cylindracea
), Amano N (
Rhizopus niveus
), Amano R (Penicillium sp.), Amano FAP (
Rhizopus oryzae
), Amano AP-12 (
Aspergillus niger
), Amano MAP (
Mucor meihei
), Amano GC-4 (
Geotrichum candidum
), Sigma L-0382 and L-3126 (porcine pancreas), Lipase OF (Sepracor), Esterase 30,000 (Gist-Brocarde), KID Lipase (Gist-Brocarde), Lipase R (Rhizopus sp., Amano), Sigma L-3001 (Wheat germ), Sigma L-1754 (
Candida cylindracea
), Sigma L-0763 (
Chromobacterium viscosum
) and Amano K-30 (
Aspergillus niger
). Enzymes identified by Patel include enzymes derived from animal tissue such as esterase from pig liver, &agr;-chymotrypsin and pancreatin from pancreas such as Porcine Pancreatic Lipase (Sigma). While these enzymes may be used in the stereoselective hydrolysis of &bgr;-lactams, the required purification of the enzyme can significantly increase the cost of the preparation of the &bgr;-lactam.
Whitesell et al. (Whitesell, J. K. Lawrence, R. M.
Chimia
, 1986, 40, 315) and Basavaiah et al. (Basavaiah,, D. and Rao, P.
Tetrahedron. Asym
., 1994, 5, 223-234) reported successful application of pig liver acetone powder (PLAP), bovine liver acetone powder (BLAP) and chicken liver acetone powder (CLAP), in the resolution of numerous chiral secondary alcohols. Experimental evidence obtained to date, however, suggests that the use of these materials results in a product having relatively low optical purity. While the reason for this is not entirely clear, it is believed that this is the result of incomplete reaction rather than the enzyme's lack of selectivity which, in turn, is likely a consequence of inconsistent amounts of active enzyme present in different batches of the liver acetone powder.
SUMMARY OF THE INVENTION
Among the objects of the present invention, therefore, is the provision of enzymatic processes for the resolution of enantiomeric mixtures of &bgr;-lactams useful in the preparation of taxanes which offers improved reproducibility as compared to processes which employ acetone powders of animal livers and which compares favorably in cost to processes which employ purified lipases and other enzymes.
Briefly, therefore, the present invention is directed to a process for the resolution of a racemic mixture of &bgr;-lactams which contain an ester. The process comprises selectively hydrolyzing the ester of one of the enantiomers by combining the mixture with homogenized liver.
Other objects and features of this invention will be in part apparent and in part pointed out hereinafter.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. Starting Materials
In general, the &bgr;-lactam enantiomers in the mixture have the following structural formula:
wherein
X
1
is —OX
6
;
X
2
is hydrogen, hydrocarbyl, substituted hydrocarbyl, or heterocyclo;
X
3
is hydrogen, hydrocarbyl, substituted hydrocarbyl, or heterocyclo;
X
4
is hydrocarbyl, substituted hydrocarbyl, or heterocyclo;
X
5
is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX
10
, —COOX
10
, or —CONX
8
X
10
;
X
6
is acyl;
X
8
is hydrogen, hydrocarbyl, substituted hydrocarbyl, or heterocyclo; and
X
10
is hydrocarbyl, substituted hydrocarbyl, or heterocyclo.
Preferably, X
2
and X
3
are hydrogen and the mixture contains the 3S,4R and 3R,4S enantiomers, and more preferably a racemic mixture of these enantiomers. Still more preferably, X
2
and X
3
are hydrogen; X
5
is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX
10
, or —COOX
10
; X
10
is alkyl, aryl or heterocyclo; and the mixture contains the 3S,4R and 3R,4S enantiomers, preferably a racemic mixture of these enantiomers. In a particularly preferred embodiment, X
2
and X
3
are hydrogen; X
5
is hydrogen, hydrocarbyl, substituted hydrocarbyl, —COX
10
, or —COOX
10
; X
10
is alkyl, aryl or heterocyclo; X
4
is 2-furyl, 3-furyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, substituted phenyl (substituted with any of the substituents identified elsewhere herein as hydrocarbyl substituents such as halo or nitro with the phenyl being mono or poly substituted in one or more of the ortho, meta or para positions), cycloalkyl, or alkenyl, and the mixture contains the 3S,4R and 3R,4S enantiomers, preferably a racemic mixture of these enantiomers.
Enantiomeric mixtures of the &bgr;-lactam starting materials may be obtained as described in Example 1 herein, or by methods analogous to those described in U.S. Pat. No. 5,229,526 which is incorporated herein by reference.
Homogenates of fresh or fresh frozen crude liver may be prepared using a high speed blender or other grinder. The liver is ground into pieces of a relatively small size and suspended in an aqueous solution. Preferably, about 1 pound (about 450 grams) of liver is combined with sufficient liquid to form about 0.5 to about 2 liters of homogenate, more preferably about 0.75 to about 1.25 liters, and most preferably about 1 liter of homogenate. In a preferred embodiment, the homogenate is buffered, preferably to a pH of about 8 with a phosphate or other suitable buffering agent.
Derivatives of homogenates are also included within the invention, such as refined fractions. To obtain refined fractions one may subject the homogenate to a series of fractionation procedures, the number of fractionation steps employed being dependent on the degree of purification desired. The series of fractionation steps could involve column chromatography such as a gel filtration column, centrifugation, heat treatment, precipitation, filtration or various other appropriate means of purification.
In general, the liver may be obtained from any animal. Preferably, the liver is avian or mammalian, more preferably chicken, turkey, pig or beef, and most preferably beef.
Stereoselective Hydrolysis
In general, a solution of &bgr;-lactam mixture in an organic solvent is combined with the homogenate to form a reaction mixture which contains about 1 gram of &bgr;-lactam to about 5 ml. to about 100 ml. of homogenate, more preferably about 1 gram of &bgr;-lactam to about 50 ml. of homogenate (with the homogenate containing about 450 grams of liver and sufficient liquid to form about 1 liter of homogenate). The reaction mixture is preferably adjusted to and maintained at about pH 7 to pH 8, preferably with a buffer, more preferably with a phosphate buffer.
The hydrolysis is preferably conducted in an aqueous, such as a buffered aqueous (e.g., phosphate buffer), medium or in an aqueous medium containing a miscible
Holton Robert A.
Vu Phong
FSU Research Foundation, Inc.
Saucier Sandra E.
Senniger Powers Leavitt & Roedel
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